Information encoded within linear segments of DNA (genes) is the basis for all life on planet Earth. Genes are not "on" all of the time - on the contrary, the activities of individual genes are finely tuned in response to various internal and external stimuli. Regulation of gene expression is critical for almost every biological process in humans and all other organisms. Small regulatory RNAs termed microRNAs have recently been discovered to be major components of gene expression control in both animals and plants. The regulation of specific "target" genes by microRNAs is critical for embryonic development, organogenesis, and stress responses. A core set of plant microRNAs regulate homologous targets in very different plant species;these ancient microRNA-target interactions are thought to regulate developmental processes. How do the same microRNA-target interactions guide the very different developmental patterns and morphologies of highly divergent plants? Do they ultimately control ancient, conserved suites of gene expression, or have they been differentially utilized in different lineages? This project seeks to answer these questions by using molecular genetics to elucidate the functions and regulatory networks of ancient miRNAs in two very different land plants. Specifically, this project will 1) genetically dissect the functions of the microRNA machinery in the moss P. patens, 2) analyze the functions of homologous, ancient microRNA-target interactions in two very different model plant species, P. patens and the flowering plant Arabidopsis thaliana, and 3) empirically determine and compare the gene regulatory networks controlled by ancient microRNA-target interactions in these two species. This project also will implement a novel methodology for the study of microRNA functions with the potential for broad applicability in basic and applied research. A detailed molecular picture of the evolution of critical gene regulatory networks will emerge from this work. Importantly, the core mechanisms of microRNA function are conserved between plants, animals, and humans. Indeed, the basic knowledge gained from past research on microRNAs and other small RNAs in easily studied plants has directly contributed to advances in human health research, including the use of RNA-interference to rapidly assay gene function in human cells, the growing appreciation of the roles microRNAs play in various common diseases, and the development of small RNA-based therapies for specific ailments. Further advances in the basic understanding of microRNA-controlled gene regulatory networks, derived by exploiting the powerful molecular genetic tools uniquely available in plant systems, will certainly continue to inform a wide variety of biomedical research. PUBLIC HEALTH RELEVANCE: Project Narrative Many genes have recently been shown to be controlled by microRNAs, including several implicated in cancer and other diseases, but the molecular details of microRNA functions are still largely unknown. Fundamental aspects of microRNA function appear identical in humans, animals, and plants, which allows analyses in easily manipulated organisms to inform human processes. This project will reveal basic mechanisms of microRNA-mediated gene control by defining the roles of ancient microRNAs in readily studied model organisms.